Cholesterol circulates through the body predominantly as components of lipoprotein particles (lipoproteins), which are composed of a protein portion consisting of one or more apolipoproteins (Apo) and various lipids, including phospholipids, triacylglycerols (triglycerides), cholesterol and cholesteryl esters. There are ten major classes of apolipoproteins: Apo A-I, Apo A-II, Apo-IV, Apo B-48, Apo B-100, Apo C-I, Apo C-II, Apo C-III, Apo D, and Apo E.
Lipoproteins are classified by density and composition. High density lipoproteins (HDL), one function of which is to mediate transport of cholesterol from peripheral tissues to the liver, have a density usually in the range of approximately 1.063-1.21 g/ml. HDL contain various amounts of Apo A-I, Apo A-II, Apo C-I, Apo C-II, Apo C-III, Apo D, Apo E, as well as various amounts of lipids, such as cholesterol, cholesteryl esters, phospholipids, and triglycerides.
In contrast to HDL, low density lipoproteins (LDL), which generally have a density of approximately 1.019-1.063 g/ml, contain Apo B-100 in association with various lipids. In particular, the amounts of the lipids, cholesterol, and cholesteryl esters are considerably higher in LDL than in HDL, when measured as a percentage of dry mass. LDL are particularly important in delivering cholesterol to peripheral tissues.
Very low density lipoproteins (VLDL) have a density of approximately 0.95-1.006 g/ml and also differ in composition from other classes of lipoproteins, both in their protein and lipid content. VLDL generally have a much higher amount of triglycerides than do HDL or LDL and are particularly important in delivering endogenously synthesized triglycerides from liver to adipose and other tissues.
Even less dense than LDL, chylomicrons (density usually less than 0.95 g/ml) contain Apo A-I, Apo A-II, Apo B, Apo C-I, Apo C-II, and Apo C-III and mediate transport of dietary triglycerides and cholesteryl esters from the intestine to adipose tissue and the liver.
The features and functions of the various lipoproteins have been extensively studied. (See, for example, Mathews, C. K. and van Holde, K. E., Biochemistry, pp. 574-576, 626-630 (The Benjamin/Cummings Publishing Co., Redwood City, Calif., 1990); Havel, R. J. et al., “Introduction: Structure and metabolism of plasma lipoproteins”, in The Metabolic Basis of Inherited Disease, 6th ed., pp. 1129-1138 (Scriver, C. R., et al., eds.)(McGraw-Hill, Inc., New York, 1989); Zannis, V. I., et al., “Genetic mutations affecting human lipoproteins, their receptors, and their enzymes”, in Advances in Human Genetics, Vol. 21, pp. 145-319 (Plenum Press, New York, 1993)).
Decreased susceptibility to cardiovascular disease, such as atherosclerosis, has been generally correlated with increased absolute levels of circulating HDL and also with increased levels of HDL relative to circulating levels of lower density lipoproteins such as VLDL and LDL (see, for example, Gordon, D. J., et al., N. Engl. J. Med., 321: 1311-1316 (1989); Castelli, W. P., et al., J. Am. Med. Assoc., 256: 2835-2838 (1986); Miller, N. E., et al., Am Heart J., 113: 589-597 (1987); Tall, A. R., J. Clin. Invest., 89: 379-384 (1990); Tall, A. R., J. Internal Med., 237: 5-12 (1995)).
Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl esters from HDL to triglyceride-rich lipoproteins such as VLDL and LDL, and also the reciprocal exchange of triglycerides from VLDL to HDL (Tall, A. R., J. Internal Med., 237: 5-12 (1995); Tall, A. R., J. Lipid Res., 34: 1255-1274 (1993); Hesler, C. B., et al., J. Biol. Chem., 262: 2275-2282 (1987); Quig, D. W. et al., Ann. Rev. Nutr., 10: 169-193 (1990)). CETP may play a role in modulating the levels of cholesteryl esters and triglycerides associated with various classes of lipoproteins. A high CETP cholesteryl ester transfer activity has been correlated with increased levels of LDL-associated cholesterol and VLDL-associated cholesterol, which in turn are correlated with increased risk of cardiovascular disease (see, for example, Tato, F., et al., Arterioscler. Thromb. Vascular Biol., 15: 112-120 (1995)).
Hereinafter, LDL-C will be used to refer to total cholesterol, including cholesteryl esters and/or unesterified cholesterol, associated with low density lipoprotein. VLDL-C will be used to refer to total cholesterol, including cholesteryl esters and/or unesterified cholesterol, associated with very low density lipoprotein. HDL-C will be used to refer to total cholesterol, including cholesteryl esters and/or unesterified cholesterol, associated with high density lipoprotein.
All lipoproteins contain apolipoproteins that serve to maintain the structural integrity of lipoproteins and mediate the transport and metabolism of lipids by acting as ligands for specific receptors or co-factors of certain enzymes. In addition to CETP, other proteins, including hepatic lipase, lipoprotein lipase, lecithin:cholesterol acyltransferase (LCAT), LDL receptor, HDL-receptor (SR-B1) and chylomicron remnant receptor, are important in lipid transport and metabolism. Disruption in the function of these components may lead to dyslipidermia, the abnormal metabolism of plasma lipids, which in turn may contribute to the development of atherosclerosis.
The proteins, apolipoproteins, and lipoproteins described above participate in three pathways of lipid transport and metabolism: (1) the chylomicron pathway, (2) the VLDL-LDL pathway, and, (3), the reverse cholesterol pathway. Chylomicrons and chylomicron remnants transport dietary lipids from intestine to peripheral tissues, such as adipose tissue, and the liver. The VLDL-LDL pathway transports lipids from the intestine to peripheral tissues. In the reverse cholesterol pathway excess cholesterol, which cannot be degraded by most tissue, is esterified and delivered either directly in HDL or indirectly after exchange into other lipoprotein fractions to the liver for excretion from peripheral tissues. Specifically, nascent HDL, which is produced by the liver and intestine, enlarges and is transformed into HDL3 and then to HDL2 as cholesterol is acquired and esterified to cholesteryl ester. Cholesteryl esters (CE) can remain with HDL2 for transport and uptake by the liver or can be transferred to lower density lipoproteins, such as VLDL and LDL, by CETP in exchange for triglycerides. In the liver, HDL2 is depleted of triglycerides by hepatic lipase which converts HDL2 back to HDL3 for re-use. During this process CE may also be transferred to hepatocytes. In addition, some HDL may be directly taken up by hepatocytes (see, for example, Havel, R. J., et al., The Metabolic Basis of Inherited Disease, 6th ed., pages 1129-1138 (Scriver, C. R., et al., eds.)(McGraw-Hill, Inc., New York, 1989); Fielding, C. J., et al., J. Lipid Res., 36: 211-228 (1995)).
Thus, the transfer of CE follows one of two pathways. First, lipoproteins may deliver cholesteryl esters to the liver for excretion, thus participating in the reverse cholesterol transport pathway. Second, cholesteryl esters may be recycled back to peripheral tissues.
When all components of these pathways are operating properly, dietary lipids are rapidly absorbed, transported, and stored or utilized. In the fasting state, lipids are efficiently transported to tissue, and cholesterol is recycled or excreted. Naturally occurring dyslipidermias, perhaps as a result of mutations of apolipoproteins, are often due to dysfunction of one or several of the components in the pathways described above (see, for example, Farmer, J. A. et al., Heart Disease. A Textbook of Cardiovascular Medicine, 4th ed., pp. 1125-1160 (Braunwald, E., ed.) (W.B. Saunders Co., Philadelphia, 1992); Havel, R. J., et al., 1992; Zannis, V. I., et al, 1993). Chronic dietary excess of cholesterol may overwhelm normal mechanisms of cholesterol clearance from peripheral tissues, and atherosclerosis may result as evidenced by the development of lesions and blockage of blood flow in cardiovascular tissue.
A number of in vivo studies utilizing animal models or humans have indicated that CETP activity can affect the level of circulating cholesterol-containing HDL. Increased CETP-mediated cholesteryl ester transfer activity can produce a decrease in HDL-C levels relative to LDL-C and/or VLDL-C levels, which in turn is correlated with an increased susceptibility to atherosclerosis. For instance, injection of partially purified human CETP into rats (which normally lack CETP activity), was shown to result in a shift of cholesteryl ester from HDL to VLDL, consistent with CETP-promoted transfer of CE from HDL to VLDL (see, Ha, Y. C., et al., Biochem. Biophys. Acta, 833: 203-211 (1985); Ha, Y. C., et al., Comp. Biochem. Physiol., 83B: 463-466 (1986); Gavish, D., et al., J. Lipid Res., 28: 257-267 (1987)). In addition, transgenic mice expressing human CETP were reported to exhibit a significant decrease in the level of cholesterol associated with HDL (see, for example, Hayek, T., et al., J. Clin. Invest., 90: 505-510 (1992); Breslow, J. L., et al., Proc. Natl. Acad. Sci. USA, 90: 8314-8318 (1993)). Furthermore, whereas wild-type mice are normally highly resistant to atherosclerosis (Breslow, J. L., et al., Proc. Natl. Acad. Sci. USA, 90: 8314-8318 (1993)), transgenic mice expressing a simian CETP were reported to have an altered distribution of cholesterol associated with lipoproteins, namely, elevated levels of LDL-C and VLDL-C and decreased levels of HDL-C (Marotti, K. R., et al., Nature, 364: 73-75 (1993)). Such transgenic mice expressing simian CETP also were more susceptible to dietary-induced severe atherosclerosis compared to non-expressing control mice and developed lesions in their aortas which were significantly larger in area than found in control animals and more typical of those found in atherosclerosis (Marotti et al., 1993). Intravenous infusion of anti-human CETP monoclonal antibodies (Mab) into hamsters and rabbits inhibited CETP activity in vivo and resulted in significantly increased levels of HDL-C levels, decreased levels of HDL-triglycerides, and increased HDL size, again implicating a critical role for CETP in the distribution of cholesterol in circulating lipoproteins (see, Gaynor, B. J., et al., Atherosclerosis, 110: 101-109 (1994)(hamsters); Whitlock, M. E., et al., J. Clin. Invest., 84: 129-137 (1989)(rabbits)).
The role of CETP activity has also been studied in humans. For example, in certain familial studies in Japan, individuals that were homozygous for non-functional alleles of the CETP gene had no detectable CETP activity. Virtually no artherosclerotic plaques were exhibited by these individuals, who also showed a trend toward longevity in their families (see, for example, Brown, M. L., et al., Nature, 342: 448-451 (1989); Inazu, A., et al., New Engl. J. Med., 323: 1234-1238 (1990); Bisgaier, C. L., et al., J. Lipid Res., 32: 21-23 (1991)). Such homozygous CETP-deficient individuals also were shown to have an anti-atherogenic lipoprotein profile as evidenced by elevated levels of circulating HDL rich in cholesteryl ester, as well as overall elevated levels of HDL, and exceptionally large HDL, i.e., up to four to six times the size of normal HDL (Brown, M. L., et al., 1989, supra at p. 451).
The above studies indicate that CETP plays a major role in transferring cholesteryl ester from HDL to VLDL and LDL, thereby altering the relative profile of circulating lipoproteins to one that is associated with an increased risk of cardiovascular disease (i.e., decreased levels of HDL-C and increased levels of VLDL-C and LDL-C). Marotti et al. (Nature, 364: 73-75 (1993)) interpreted their data as indicating that a CETP-induced alteration in cholesterol distribution was the principal reason that arterial lesions developed more rapidly in transgenic, CETP-expressing mice than in non-transgenic control mice when both groups were fed an atherogenic diet.
CETP isolated from human plasma is a hydrophobic glycoprotein having 476 amino acids and a relative molecular weight of approximately 66,000 to 74,000 daltons on sodium dodecyl sulfate (SDS)-polyacrylamide gels (Albers, J. J., et al., Arteriosclerosis, 4: 49-58 (1984); Hesler, C. B., et al., J. Biol. Chem., 262: 2275-2282 (1987); Jarnagin, S. S., et al., Proc. Natl. Acad. Sci. USA, 84: 1854-1857 (1987)). A cDNA encoding human CETP has been cloned and sequenced (Drayna, D., et al., Nature, 327: 632-634 (1987)). CETP has been shown to bind cholesteryl esters (CE), triglycerides (TG), phospholipids (Barter, P. J. et al., J. Lipid Res., 21: 238-249 (1980)), and lipoproteins (see, for example, Swenson, T. L., et al., J. Biol. Chem., 264: 14318-14326 (1989)). More recently, the region of CETP defined by the carboxyl terminal 26 amino acids, and in particular amino acids 470 to 475, has been shown to be especially important for neutral lipid binding involved in neutral lipid transfer (Hesler, C. B., et al., J. Biol. Chem., 263: 5020-5023 (1988)), but not phospholipid binding (see, Wang, S., et al., J. Biol. Chem., 267: 17487-17490 (1992); Wang, S., et al., J. Biol. Chem., 270: 612-618 (1995)).
It follows from current research that increased levels of CETP activity may be predictive of increased risk of cardiovascular disease. Endogenous CETP activity is thus an attractive therapeutic target for modulating the relative levels of lipoproteins to prevent or inhibit the development of or to promote regression of cardiovascular diseases such as atherosclerosis.
It would be useful, therefore, to develop the means and methods to control or modulate endogenous CETP activity to prevent or treat cardiovascular disease. Preferably, the modulation of endogenous CETP activity in a human or animal would be accomplished by administering to the subject a pharmaceutical composition that is specific for CETP, does not require large quantities, does not require continuous or frequently repeated dosing, and also does not produce untoward side effects.